Directed evolution is the introduction and identification of new sequence differences in genes and gene products to expand their usefulness. The use of procedures to introduce heterology and to recombine and reassort DNA gene sequences for the purpose of producing novel forms and activities of the products of those genes has taken several forms, each with its advantages and disadvantages. Several in vitro (test-tube) methods have been described, including those that use insertion of randomized oligonucleotides into genes (Suzuki, Baskin, Hood, & Loeb, 1996), artificial exon shuffling of natural variants of genes by recombinant DNA methods, random mutagenesis by error-prone PCR (Cadwell & Joyce, 1993), and sexual PCR methods (Crameri, Bermudez, Raillard, & Stemmer, 1998; Stemmer, 1994; Zhang, Dawes, & Stemmer, 1997), as disclosed in Stemmer et al. in U.S. Pat. No. 5,811,238, issued Sep. 22, 1998, and Minshull et al., U.S. Pat. No. 5,837,458, issued Nov. 17, 1998.
All of the above methods of in vitro-manipulated sequences have the drawback that they require subsequent introduction of the altered sequences into a test cell for subsequent protocols, such as selection and/or screening. Since large populations of newly altered sequences are required in order to find the rare instances of beneficially altered properties, large numbers of transformations are necessary to introduce each altered sequence into a cell where the effects of the alterations can be gauged. This difficulty is frequently compounded in cases where multiple rounds of mutagenesis and reassembly, and subsequent retransformation, are used.
In vivo diversification of genes using homologous melotic recombination of heterologous genes, as disclosed in Catcheside et. al. in U.S. patent application Ser. No. 08/977,171 (Reagents and Methods for Diversification of DNA, filed November, 1997), greatly reduces the number of transformations of DNA molecules into cells that are required. This method, however, requires initial sequence differences between the two genes that are the partners in the recombination event in order to achieve new combinations of sequence. This limits the method to diversification of genes that have existing nucleotide differences, such as between natural gene variants found in related species, or molecules that have been diversified by one of the aforementioned in vitro methods. Also, the homologous recombination machinery that recombines the two subject genes is limited to sequences with relatively high levels of sequence identity. Generally, recombination is limited to the range of 1-5% heterology (Harris, Rudnicki, & Haber, 1993). Most critically, single polynucleotide sequences without close homologues cannot be diversified by this method.
The present invention advances the previous work by providing an effective means to diversify polynucleotide sequences in vivo without the need for large numbers of transformation steps. The methods and compositions of the invention are useful in the production of new gene products, metabolites, and phenotypes as well as providing information on the structure/function relationship of the products encoded by a polynucleotide sequence.